An automatic system is needed which works with a continuous or sequential sample flow and is capable of highly accurate results for the diagnostic testing of large numbers of people for the incidence of pathological amounts of uric acid in their blood. Uric acid is the end product of purine degradation in humans and is therefore found physiologically in blood. In numerous diseases, there is an increase or decrease in the uric acid concentration and therefore the determination of a variation from the normal physiological uric acid level in blood is an extremely important diagnostic indication for the physician.
Increases in the uric acid concentration are found, for example, in gout, chronic pneumonia with extensive destruction of tissue and pernicious leukemia. Decreases in uric acid concentration are found, for example, in the toxic lesions of the renal tubuli in Wilson's disease.
It is therefore important to have available an automated method for the determination of uric acid which is not only simple to carry out but which is also accurate and which can serve as an adjunct to routine screening examinations in clinics or to periodic screening examinations of cases in hospitals or nursing homes and the like.
The known methods for the determination of uric acid can be broadly classified as enzymatic, alkaline phosphotungstate and miscellaneous chemical colorimetric methods. The enzymatic method utilizing the enzyme uricase suffers the disadvantage that the measurement is carried out in the ultraviolet region. Such methods require the use of expensive quartz cuvettes and a spectrophotometer. In the alkaline phosphotungstate method the measurement is carried out in the visible region, but this method has the disadvantage that other components which act in a reductive manner are detected at the same time, thus producing values which are too high and, at the same time, incorrect.
An especially interesting method for the enzymatic determination of uric acid has been described recently by G. F. Domagk and H. H. Schlicke in Analytical Biochemistry 22, 219-224 (1968). The Domagk et al. method combines the advantage of specific enzymatic determination with the advantage of the ability to carry out the measurement in the visible region. According to this method, uric acid is converted in a first step using uricase into allantoin and hydrogen peroxide. ##STR1##
In the second step a color forming indicator reagent (chromogen) is introduced. The hydrogen peroxide resulting from the first step oxidizes the chromogen which is present in the leuco-form to a colored substance, under the catalytic action of peroxidase. o-Dianisidine is such a chromogen. ##STR2## One of the disadvantages of this method is, however, the time-consuming deproteinization step required before the determination is actually carried out. This deproteinization method is described for a manual procedure and is not suitable for automation according to the procedures of D. Susic and P. Scheibe, Z. Anal. Chem. 257, 130-132 (1971).